The overall objective of this project is to investigate the mechanisms involved in the onset of malignant transformation by chemical carcinogens. This project specifically aims to study the relationship between carcinogenesis and DNA replication. We have proposed to purify and characterize the replication complexes from regenerating rat livers and to extend the same methodology to cells in culture. The observation that DNA at the replication complex is preferentially alkylated by chemical carcinogens has been confirmed by the treatment of C3H 10T1/2 cells with N-methyl-nitrosourea. Our data has demonstrated that the DNA being replicated during the carcinogen treatment is 3-5 fold more susceptible to alkylation than the bulk DNA. We are investigating the reasons for this greater specificity for the replication fork region to methylation. We are also interested in determining if this specificity is a characteristic of carcinogens introducing a small modification into the DNA or if it is a more general phenomenon. With the help of specific antibodies and electron microscopic techniques, the relationship of carcinogen binding sites to DNA replication forks is being investigated in situ. Once the distribution of alkylating sites in DNA is determined for several carcinogens, the effects on that distribution of low versus higer more toxic doses will be studied. The specific nucleotide sites of alkylation will also be compared for DNA at and away from the replication fork. From these studies we may obtain further information on the structure of the replication fork in the presence of damaged templates and therefore, more insight about post-replication repair in 10T1/2 cells or regenerating rat liver.